1. Field of the Invention
The present invention relates to short chain neurotoxins from Lapemis hardwickii and their encoding genes.
2. Description of Related Art
Snake neurotoxins are important components of snake venom, widely existing in elapidae and sea snakes. According to their different binding targets, snake neurotoxins can be divided into two main types: presynaptic and postsynaptic neurotoxins (Singh, B. R., Tu, A. T. Overview of snake venom, In: Natural Toxin 2: Structure, Mechanism of Action and Detection. New York: Plenum Press, 1996, 391: 37˜62). Presynaptic neurotoxins inhibit acetylcholine release from presynaptic motor nerve ending, and Postsynaptic neurotoxins block nerve conduction by specifically binding to acetylcholine receptors of nicotinic amide type on postsynaptic membrane motor end-plates from vertebrates muscle or electric organs from fishes, thus influence subsequent physiological processes associated with acetylcholine of nicotinic amide type binding to motor end-plates of muscle cells (Toru, T., Satoshi, O., Eisaku, N., et al. Complete nucleotide sequences of cDNAs encoding long chain α-neurotoxins from sea krait, Laticauda semifasciata. Toxicon. 1999, 37: 181˜185; Qin, G. P. China poisonous snake research: 2nd edition. Guangxi science and technology press. 1998, 372˜385). On the basis of molecular weight and the number of inter-chain disulfide bonds, postsynaptic neurotoxins can be subdivided into two groups: short chain and long chain neurotoxins. The former consist of 60 to 62 amino acid residues with four pairs inter-chain disulfide bonds whereas the latter have 66 to 79 amino acid residues with five pairs inter-chain disulfide bonds. The two groups share a common structure characteristic of three-finger loops (Louis, W. C., Robert, S. D. Handbook of Neurotoxicology, New York: Marcel Dekker Inc, 1995, 637˜665.).
The predominant clinical features of neurotoxic symptom arise from postsynaptic neurotoxins include: flaccid muscle paralysis in one hour or even sooner, dyspraxia followed by dyspnea, ultimately leading to asphyxiant convulsive death. Artificial breathing may help delay death. Some may even get saved.
Postsynaptic neurotoxins are widely used in the realm of biology and medicine, and of especial importance when applied to molecular biology and molecular pharmacology. Analgesics made from snake neurotoxins can be used to treat rheumatic arthralgia, trifacial neuralgia, sciatica, intercostal neuralgia, swelling pain of late cancer, leprotic neuralgia and so on. The advantages thereof are manifested in rapid and effective analgesia, no tolerance after successive medication, no addiction, low dosage, generally no serious toxic side-effect. Noticeable, its application to the treatment of swelling pain from late cancer is of great significance (Chen, R. Z., et al. Analgesia of cobrotoxin. China pharmacology bulletin. 1998, 4(2): 113˜117; Hao, W. X., Chen, Y. C edited. Biochemistry, toxicology and application of snake venom. Science Press: Beijing. 1980, 109˜115.). Binding to acetylcholine receptor of nicotinic amide type with high affinity and specificity, postsynaptic neurotoxins may be used to study on neuroreceptors. Some of its application are as follows: a label to detect acetylcholine receptor of nicotinic amide type; to help purify acetylcholine receptor of nicotinic amide type; a tool to study interaction between drugs and receptors; a tool to help investigate the structure and character of acetylcholine receptor of nicotinic amide type. In addition, postsynaptic neurotoxins are especially useful with regard to study on myasthenia gravis. The main clinical symptom of myasthenia gravis is lassitude of striated muscle at slight motion, results from destruction of acetylcholine receptor of nicotinic amide type by autoimmunity. So far, a method of radioactive-iodine labeled neurotoxin and acetylcholine receptor of nicotinic amide type has been employed to test the content of antibody against acetylcholine receptor in patient's serum so as to diagnose the disease. To further study its pathogenic mechanism, short chain neurotoxins are also used to purify subunits of acetylcholine receptor of nicotinic amide type and prepare monoclonal antibody against receptor subunits. This monoclonal antibody can be used to neutralize acetylcholine receptor in animals to probe into a new therapy pathway (China poisonous snake research Qin, G. P. edited: 2nd edition. Guangxi science and technology press. 1998, 495˜497).
Production of snake neurotoxin by genetic engineering hold brilliant future, considering the fact that at present snake neurotoxins are mainly obtained by biochemical extraction from snake venom with high cost and low purity. Since the year of 1995 when the gene encoding toxic protein of Dendroaspis angusticeps was cloned and expressed (Leonard, A. S., Mark, A. O., Pierre, J. L., et al. Cloning and expression of mamba toxins. Toxicon, 1995, 33(4):439˜474.), many reports have come out about the production of snake neurotoxins or other toxic polypeptides by biological engineering (Fatemeh, A., Arunmozhiarasi, A., Nget, H. T., et al. Four new postsynaptic neurotoxins from Naja Naja Sputatrix venom: cDNA cloning, protein expression, and phylogenetic analysis. Toxincon. 1998, 36(12):1871˜1885; Nanling, G., Arunmozhiarasi, A. and Kandiah, J. Postsynaptic short-chain neurotoxins from Pseudomaja texilis cDNA cloning, expression and protein characterization. Eur. J. Biochem. 1999, 265: 982˜989.).
Sea snakes are widely distributed in the warmer drainage area of the Indian Ocean and the Pacific Ocean, and the majority live along the coast of South Asia and East Indian Ocean. They are easily recognized for their level and oar-shaped tail. Sea snake venoms, with exception of that from Emydocephalus annulatus, are highly toxic and even more intense than those from land snakes. Yet compositions of sea snake venoms are more simple than those of land snakes (Chikahisa Takasaki, 1998, The Toxinology of Sea Snake Venoms, J. Toxicol.—Toxin Reviews, 17(3): 361˜372.). Snake venom neurotoxin is the most toxic component of sea snake venom, which may lead to flaccid paralysis and respiratory failure, and consequently lead to death. Studies on sea snake toxin are much less than those of land snakes at home and abroad. Most of them rest on the protein level. Not many studies are reported on genes encoding sea snake toxins. Up to now, about over 100 amide acid sequences of sea snake toxins have been reported in Genbank whereas no more than 50 nucleotide sequences encoding sea snake toxins can be found.
Because of the intense toxicity and trace expelling, it is difficult to acquire sufficient venom from sea snakes. Thus some difficulties may arise in isolation and purification of compositions of sea snake venom, studies on structure and characterization of toxin and enzyme molecules, as well as in research and application (Marine life toxin research. Song, J. J., Mao, Q. W. edited. Beijing science and technology press: Beijing. 1996, 337˜354).
With rapid progress of research on native toxins, the clinical applications have been widely made, considering the highly specific bioactivity that native toxins possess, snake venom obviously takes a predominant position as to the application of toxin. Scientists try to utilize native bioactive substance as an important guide looking for new drugs. The utilization of snake venom has drawn close attention from researchers on pharmacology, medicament, biochemistry and medicine and gained rapid development in the application field. Neurotoxins are mainly found in venoms of snakes belonging to the sea snake family and elapidae, and have a long history of application to analgesia. The first relevant report was from Macht in 1936. Steinbrocker utilized cobrotoxin to treat 65 patients with arthritis or neuralgia in 1940 and got an effective rate up to 59%. The three companies Hynson, Westco and Dunning took advantage of snake venom to produce two analgesics Cobroxin and Nyloxin. The former is neurotoxin, with 1.0 ml per ampule and 50×106 IU in content, directed against intractable pain and carcinoma pain. The latter, with 3.3×106 IU of neurotoxin added by 1.5 ml of silicic acid and 3.3 ml of formic acid to form physiological saline, is targeted at arthralgia. These two drugs are on list of American Bureau of Drug. In 1975, Kunming Zoology Institute of China Science Academy successfully developed cobrotoxin injection using cobrotoxin. The injection has been put to batch manufacture by Wuzhou Pharmaceutical Factory in Guangxi province, commercially designated as “Ketongning Injection”. It is one novel analgesic, characteristic of rapid and effective analgesia, no tolerance after successive medication, no addiction, low dosage and no serious side-effect. It can be used to treat rheumatic arthralgia, trifacial neuralgia, sciatica, intercostal neuralgia, swelling pain of late cancer, leprotic neuralgia and so on. But the drug is restricted to intramuscular injection and it will take 3 to 5 days to take effect accompanied with sclerotic swelling at injection site. Subsequently, snake venom clinical application research center of PLA employed modern techniques to isolate and purify cobrotoxin and prepared one new analgesic named “New Ketongning” which takes effect immediately by either intravenous or intramuscular injection. Cobrotoxin has been used to relieve carcinoma pain, various neuralgia, arthralgia for over 60 years at home and abroad (Chen, R. Z., Wu, X. R. Analgesia of cobrotoxin. China pharmacology bulletin. 1988, Vol: 4, Issue: 2). Cobrotoxin is applied to clinical treatment in many internal hospitals by intravenous or intramuscular injection, and prove to be safe in a dosage of 1/2500th of LD50 of mice {one dosage each day, 1˜2 bottle each time, for particular two dosages each day and 1 bottle each time. As for acute pain, withdrawal on abolition of pain. As for chronic pain, 3˜5 more administrations for consolidation. 20 days for one period of treatment and repetitive periods are needed if necessary (Ketongning, 2 ml per bottle with 70 μg neurotoxin)}, safe and reliable (Wang, X. Y/207 hospital, Siping City, Jilin Province., Wang, F. X/chief hospital of battalion, Shenyang, Analgesia observation of New Ketongning after operation. Snake record. 1999, Vol: 11, Issue: 1; Gao, Z. E/traditional Chinese medical hospital, Suhzhou City, Jiangsu Province, Analysis of 182 sciatica treatments with Ketongning matched with traditional Chinese medicine. Snake record. 1998, Vol: 10, Issue: 3; Cao, Y. S., Cheng, B. Q., Zhao, G. H., et al/Military Medical Institute of logistic administration, battalion in Guangzhou/6th people hospital, Guangzhou City., Comparation and observation of clinical analgesia of Ketongning and its compound. China Journal of biochemical drugs. 1996.) Snake neurotoxins are also applied to abstinence of drugs and obtained good treatment effect by oral administration clinically (Yang, L., Li, H., Wu, Y. W et al/treatment centre of studying on drug dependency, Kunming medical college., Clinical effect observation of snake venom capsule in treatment of Heroin addiction. Withdrawal medicine.).
Snake neurotoxins share considerable amide acid sequence homology. At present, available neurotoxins mainly come from snake venom by biochemical extraction which underlie some defects such as high cost and low purity which may always lead to medical negligence. Now snake neurotoxins produced by gene engineering technique not only can solve the above problems, but also have the same effect of medicine comparable with that of native snake neurotoxin.